EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-*Make sure the magnetic bead slurry is well mixed before removing appropriate volume for IP, as magnetic beads will settle on the bottom of the tube over time. -For user-provided antibody and controls, add between 1-10 ug of antibody per tube. -Use ChIP validated antibody.

Protocol tips
-*Make sure the magnetic bead slurry is well mixed before removing appropriate volume for IP, as magnetic beads will settle on the bottom of the tube over time. -For user-provided antibody and controls, add between 1-10 ug of antibody per tube. -Use ChIP validated antibody.

Publication protocol

Approximately 50 µg of minced liver tissue was fixed for 10 min with 1% formaldehyde to cross-link DNA and chromatin-binding proteins such as transcription factors to ensure coimmunoprecipitation (as per standard protocol using a Magna ChIP G kit obtained from Millipore, Billerica, MA). Cross-linked chromatin was neutralized by addition of excess glycine to quench unreacted formaldehyde. Chromatin was sheared by sonication to generate fragments of 200–1,000 bp size. The fragment size range was confirmed by a 1% agarose gel electrophoresis. C/EBP-α and C/EBP-β chromatin immunoprecipitation (ChIP) were performed using 1 µg cross-linked chromatin and subsequent immunoprecipitation with 2 µg antibody from Santa Cruz Biotechnology (C/EBP-α: Cat. #sc-61X and C/EBP-β: Cat. #sc-150X). The immunoselection was performed using the ChIP grade antibody in combination with protein G-conjugated solid support matrix magnetic beads to enrich for the specific DNA-protein complex of interest.

ChIP was followed by ultrahigh-throughput sequencing using the ABI SOLiD ChIP-seq platform to detect C/EBP-β and C/EBP-α binding targets in liver samples obtained at 6 h following PHx. SOLiD reads were aligned to the reference genome (Rn4) using Bioscope 1.2 with default parameters (seed of 25 bases with 2 mismatches for 50 base-pair reads). The alignment algorithm used a seed-and-extend approach similar to BLAST. The alignment was extended in both directions with a mismatch penalty of −2 and a matching score of +1, and the alignment with the highest score was reported.

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Manufacturer protocol

Download the product protocol from Merck Millipore for EZ-Magna ChIP™ G - Chromatin Immunoprecipitation Kit below.

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